The major complication associated with organ transplantation is allograft rejection, which is mediated by graft-reactive T cells. Rejection is controlled by non specific immunosuppressive therapies, which often lead to life threatening infections. Hence, transplant patients would benefit significantly from the development of therapies which selectively target graft.reactive T cells and induce graft-specific tolerance. It is widely accepted that CTL and DTH responses are the principle effector mechanisms of allograft rejection. Members of the beta-chemokine family of chemoattractants likely regulate graft infiltration by CLT and other leukocytes. Since CLT and DTH responses are promoted by the Th1 products IL-2 and IFN-gamma graft-reactive Th1 cells provide a target for tolerance inducing strategies. It is well documented that the Th2 products IL-4 and IL-10 inhibit Th1 development. Further, evidence suggests that Th2 cytokines influence chemokine production. Hence, an increasingly popular hypothesis states that selective induction of graft-reactive Th2 cells would promote graft.specific tolerance. By promoting an early shift from Th1 to Th2 cytokine production, acute rejection episodes, which likely contribute to chronic rejection, may be prevented. To date, this attractive hypothesis has not been rigorously tested. Since we and others have reported that Th2 cytokines may promote alternate (non Cit) mechanisms of graft rejection, it is essential to define the in vivo biologic activities of Th2 cells in the context of transplantation. We hypothesize that Th2 cells mediate both tolerogenic and deleterious activities and that these activities may be segregated for therapeutic purposes. This hypothesis will be tested in a comprehensive in vivo system which combines the mouse cardiac transplant model with modified LDA to quantify graft-reactive T cell development, RT-PCR to evaluate in situ cytokine gene expression, histologic analyses to evaluate tissue damage, and immunohistochemistry to evaluate endothelia expression of chemokines. Specifically, we will: l) Identify tolerogenic and deleterious activities of Th2 and determine if deleterious activities may be ablated leaving tolerance inducing factors intact. Graft-reactive Th2 will be generated in vitro and adoptively transferred into Thy l congenic and scid cardiac allograft recipients. The roles of eosinophils, macrophages, and alloantibody in Th2 mediated tissue damage will be assessed. 2) Determine if a Th1 to Th2 shift may be induced therapeutically. Two key regulatory cytokines will be manipulated as an inductive therapy. IL-12 will be neutralized in vivo, and intragraft expression IL-10 will be achieved by gene transfer into donor hearts. 3) Determine the role of beta-chemokines in recruitment of effector mechanisms. and determine if chemokine activity may be targeted to promote allograft tolerance. This information will be essential for the development of tolerance inducing therapies based on in vivo cytokine manipulation. It is important to note that several key observations made in this experimental mouse model have been verified in human cardiac transplant patients. Hence, we anticipate that the information gained from these studies will have direct clinical relevance.